Evaluation of strategies for improving the transgene expression in an oleaginous microalga Scenedesmus acutus.

BACKGROUND: Genetic transformation of microalgae has been hampered by inefficient transgene expression, limiting the progress of microalgal biotechnology. Many vector tools and strategies have been developed in recent years to improve transgene expression in the model microalga Chlamydomonas, but these were hardly applied to other microalgae. In this work, naturally-isolated oleaginous microalgae were accessed for genetic transformation, and various expression systems were evaluated in a selected microalga to circumvent inefficient transgene expression. RESULTS: Initially, a strain of Scenedesmus acutus was selected from the oleaginous microalgal collection based on its highest transformation rate and transgene stability. This strain, which had very low or no GFP reporter expression, was first tested to improve transgene expression by using intron-containing constructs and the transcript fusion using ble::E2A. The intron-containing constructs yielded 2.5-7.5% of transformants with 2-4-fold fluorescence signals, while the majority of the transformants of the transcript fusion had the fluorescence signals up to 10-fold. Subsequently, three UV-induced S. acutus mutants were isolated with moderate increases in the level and frequency of transgene expression (2-3-fold and 10-12%, respectively). Finally, a transcript fusion system was developed using psy white mutants with an expression vector containing PSY::E2A for complementation and light selection. Transformants with green colonies were selected under light exposure, and the transgene expression was detected at protein levels. Although the improvement using PSY::E2A was only minor (1-2-fold increase and ~ 7% of transformants), this system provides an alternative selectable marker that is compatible with large-scale culture. CONCLUSIONS: Here, the overall improvement of transgene expression using the Chlamydomonas tools was moderate. The most effective tool so far is the transcript fusion using ble::E2A system. This work demonstrates that, so far, genetic engineering of non-model microalgae is still a challenging task. Further development of tools and strategies for transgene expression in microalgae are critically needed.

Non-ionic surfactant-assisted controlled release of oxyresveratrol on dendritic fibrous silica for topical applications.

We present a simple and eco-friendly method for controlled drug release using a surfactant-assisted method. Oxyresveratrol (ORES) was co-loaded with a non-ionic surfactant onto KCC-1, a dendritic fibrous silica, using an ethanol evaporation technique. The carriers were characterized using FE-SEM, TEM, XRD, N2 adsorption-desorption, FTIR, and Raman spectroscopy, and the loading and encapsulation efficiencies were assessed using TGA and DSC techniques. Contact angle and zeta potential were used to determine the surfactant arrangement and the particle charges. To investigate the effects of different surfactants (Tween 20, Tween 40, Tween 80, Tween 85, and Span 80) on ORES release, we conducted experiments under different pH and temperature conditions. Results showed that the types of surfactants, drug loading content, pH, and temperature significantly affected the drug release profile. The percentage of drug loading efficiency of the carriers was in the range of 80 %-100 %, and the release of ORES was in the order of M/KCC-1 > M/K/S80 > M/K/T40 > M/K/T20 > MK/T80 > M/K/T85 at 24 h. Furthermore, the carriers provided excellent protection for ORES against UVA and maintained its antioxidant activity. KCC-1 and Span 80 enhanced the cytotoxicity to HaCaT cells, while Tween 80 suppressed the cytotoxicity.

Proteomics and Molecular Docking Analyses Reveal the Bio-Chemical and Molecular Mechanism Underlying the Hypolipidemic Activity of Nano-Liposomal Bioactive Peptides in 3T3-L1 Adipocytes.

Obesity is a global health concern. Physical activities and eating nutrient-rich functional foods can prevent obesity. In this study, nano-liposomal encapsulated bioactive peptides (BPs) were developed to reduce cellular lipids. The peptide sequence NH2-PCGVPMLTVAEQAQ-CO2H was chemically synthesized. The limited membrane permeability of the BPs was improved by encapsulating the BPs with a nano-liposomal carrier, which was produced by thin-layer formation. The nano-liposomal BPs had a diameter of ~157 nm and were monodispersed in solution. The encapsulation capacity was 61.2 ± 3.2%. The nano-liposomal BPs had no significant cytotoxicity on the tested cells, keratinocytes, fibroblasts, and adipocytes. The in vitro hypolipidemic activity significantly promoted the breakdown of triglycerides (TGs). Lipid droplet staining was correlated with TG content. Proteomics analysis identified 2418 differentially expressed proteins. The nano-liposomal BPs affected various biochemical pathways beyond lipolysis. The nano-liposomal BP treatment decreased the fatty acid synthase expression by 17.41 ± 1.17%. HDOCK revealed that the BPs inhibited fatty acid synthase (FAS) at the thioesterase domain. The HDOCK score of the BPs was lower than that of orlistat, a known obesity drug, indicating stronger binding. Proteomics and molecular docking analyses confirmed that the nano-liposomal BPs were suitable for use in functional foods to prevent obesity.

Discovery of a Multifunctional Octapeptide from Lingzhi with Antioxidant and Tyrosinase Inhibitory Activity.

Ganoderma lucidum or Lingzhi is a fungus species widely known as a traditional medicine. Exploring the beneficial peptides by hydrolysis using pepsin and trypsin has been extensively performed to identify new bioactive natural products. A multifunctional peptide that expresses potential scavenging activity and tyrosinase inhibition is valuable in therapeutic and cosmetic applications. This study aimed to identify and investigate the effects of a novel multifunctional peptide from Lingzhi on the melanogenic enzymes in melanoma cells by a targeted-proteomics approach. The multifunctional peptide was de novo sequenced by LC-MS/MS to be NH2-PVRSSNCA-CO2H (octapeptide). This sequence was chemically synthesized by solid-phase peptide synthesis (SPPS). The antioxidant ability of the synthesized octapeptide was measured by the DPPH, ABTS, and FRAP assays. The results showed that the peptide exhibited an antioxidant activity equal to 0.121 ± 0.01 mg equivalent to ascorbic acid, 0.173 ± 0.03 mg equivalent to gallic acid, and 2.21 ± 0.23 mM equivalent to FeSO4, respectively, which is comparable to these well-known antioxidants. The proteomics approach identified a total of 5804 proteins and several pathways involved in the effects of the octapeptide in melanoma cells. Targeted proteomics revealed three specific proteins associated with pigmentation including Rab29, Dct, and Tyrp1. The Rab29 and Dct were upregulated whereas Tyrp1 was downregulated in the octapeptide treatment group. These findings could be used in the understanding of the molecular functions of the multifunctional octapeptide on melanogenic enzymes, supporting its potential as a therapeutic and cosmetic ingredient.